Glucose effect on degradation kinetics of methyl parathion by filamentous fungi species Aspergilus Niger AN 400

This study evaluated the glucose effect on the removal of methyl parathion by Aspergillus niger AN400. The study was conducted in two stages: toxicity tests on plates and assays in flasks, under an agitation of 200 rpm. The methyl parathion concentrations in the toxicity test ranged from 0.075 to 60 mg/L. The second stage consisted on evaluating reactors: six control reactors with methyl parathion solution; six reactors with fungi and methyl parathion, and six reactors containing fungi, methyl parathion, and glucose. The reaction times studied ranged from 1 to 27 days. Methyl parathion concentrations of up to 60 mg/L were not toxic for Aspergillus niger AN400. The first-order kinetic model served as a good representation of the methyl parathion conversion rate. The first-order kinetic constant was 0.063 ± 0.005 h-l for flasks without addition of glucose, while a value of 0.162 ± 0.014 h-l was obtained when glucose was added.

Fungi have been employed extensively to remove toxic and recalcitrant compounds.Garcia et al. (2000) used the species Aspergillus niger, Aspergillus terreus and Geotrichum candidum in the removal of phenolic compounds; Volke-Spulveda et al. (2003) studied hexadecane biodegradation by Aspergillus niger; and Bruce et al. (1995) investigated the degradation of pentachlorophenol by the fungi species Phanerochaete chrisosporyum, Trametes versicolor and Inonotus dryophilus.
Glucose addition is important to improve the efficiency of bioremediation of persistent compounds like dyes (YANG et al., 2008;RODRIGUES et al., 2010), phenols (RODRIGUES et al., 2007;SILVA et al., 2007) and pesticides (SAMPAIO, 2005;YANG et al., 2008).Singh (2006) reports that glucose addition produces substances of high reactivity, which react more easily with the pollutant.This research focused on evaluating MP removal by Aspergillus niger in the presence and absence of glucose, and on estimating the biological degradation kinetics.
Glucose was chosen because it is a primary substratum and the main carbon source for this fungus.

Cultivation and production of the fungus species
Aspergillus niger AN400 was grown on Petri dishes at a tempera-

Introduction
The environmental contamination resulting from worldwide indiscriminate, abusive, and long-term use of pesticide is a cause of great concern to public authorities and health providers, for it seriously impacts the sustainability of natural resources and human health.
One of the consequences of the widespread use of pesticides in agriculture is the contamination of water bodies.The use of agrochemicals close to flooded areas has led to the intoxication of many fish species (ESPINDOLA et al., 2000).It represents a serious pollution problem that causes environmental imbalance and a high incidence of fish poisoning, which is harmful to aquatic and human life.
Several factors are directly related in the persistence and toxicity of these compounds in the environment, including soil and water mobility, half-life in soil and water, frequency of application, climatic conditions, and irrigation (SUDO et al., 2002).
According  , 1987).Although its activity in the environment is short-lived and little dispersive, methyl parathion (MP) can be highly toxic for humans.Toxicity by this organophosphorate results from the inhibition of the enzyme acetylcholinesterase, which causes acetylcholine to be accumulated in the body, affecting the central nervous system and sometimes leading to fatal respiratory failure (HERNANDEZ et al., 1998).Despite these hazards, this pesticide is widely used in agriculture.

Culture
Two were prepared culture media.

Assays in batch reactors
Eighteen Erlenmeyer flasks (250 mL) were used as reactors.
They were sealed and divided into sets.The first set consisted on six control reactors containing 100 mL of culture medium 1 (C), with different MP concentrations, while the second one consisted on six reactors containing 100 mL of culture medium, one with six different MP concentrations and two x 10 6 A. niger's spores (PF), and the third set contained 100 mL of culture medium 2 with six different MP concentrations and 2 x 10 6 A. niger's spores (PFG).Table 1 presents the initial MP concentrations in each reactor.
All reactors were covered with black plastic bags and subjected to 200 rpm shaking in the shaker used in the first stage.The temperature was kept at 30°C throughout the experiment.
The parameters analyzed were pH, volatile suspended solids (VSS) and MP concentrations.Analyses were performed according to APHA (1995).

Kinetic evaluation of MP degradation
The effect of glucose on the pesticide degradation rate was evaluated though kinetic studies, using temporal profiles of MP concentration for each condition under study.The initial rate (Ro) was estimated at time zero by the mass balance equation for batch reactor (Equation 1).

Ro = -dC MP dt
Equation 1 Where: C MP is the MP concentration and "t" is the time.

Analytical methods
The MP was quantified using a Shimadzu Liquid Chromatograph (LC-10 AD), which was equipped with UV-visible diode array detector (SPD-10AVP), column oven (CTO -10AS), and a low-pressure pump system (SL -10 AVP), operating with up to four solvents.
The insecticide was separated on a Supelco C18 column (25 cm x 4,6 mm D.I; 5 µm particles), under the following chromatographic conditions: isocratic system with a phase acetonitrile : water -80% (1 mL/min), an initial run time of five minutes, detection at 270 nm, and a 20 µL injection volume.
The pH was determined using a Universal Indicator of pH 0-14 paper (Merck), and the VSS were quantified according to the Standard Methods for Examination of Water and Wastewater (APHA, 1995).
The samples for analysis, collected in a sterile atmosphere provided by a Bunsen burner, were poured into sterilized Ependorff flasks.
The pH was determined at the moment of sampling, and the sample was refrigerated at 4 o C for subsequent determination of the MP concentration.The VSS concentration was determined at the end of the experiment in all samples from the PF and PFG reactors.(INGELSE et al., 2001).

Assays in batch reactors
Figure 1 shows the concentration of MP in the batch reactors (PF) over time.Clearly, Aspergillus niger was able to remove MP from the liquid phase, since all reactors inoculated with the fungus showed a drop in the MP concentration during the experiment, while the control reactors maintained the same MP concentration throughout the experiment.The inhibitory effect of MP on the removal efficiency was also clear, i.e., the reactors with initial MP concentration of 0.2 mg/L displayed a removal efficiency of 51% after 27 days, while the PF6, which is a reactor with an initial MP concentration of 19.1 mg/L, removed only 2%.
Figure 2 shows the beneficial effect on the MP removal rate resulting from the addition of glucose.The highest removal efficiency was 82%, which was achieved by the reactor with the lowest initial concentration (0.62 mg/L).Inhibition due to the insecticide was also evident, for the removal efficiency increased at a lower initial MP concentration; in other words, the highest initial concentration tested (24.89 mg/L) resulted in a removal efficiency of 43%.According to Griffin (1994), glucose presence reduces the lag phase, hastening the exponential growth phase.
The enzymatic action of the fungus may have been responsible for the degradation of MP.This fungus possesses several enzymatic systems, such as: glucose oxidase, catalase, lactanase (WITTEVEEN, 1993), cytochrome P450 monooxygenase and ligninolytic enzymes (PRENAFETA BOLDÚ, 2002).citocrome P450, the chloroperoxidase was not capable of cleaving the oxone structures.
The influence of glucose on MP degradation can also be evaluated through a kinetic study.The initial Ro values were obtained from the temporal profiles of MP concentration, resulting from several initial MP concentrations (Equation 1).The values of Ro are presented in Table 2, and data for control reactors are not shown.
The first-order kinetic model represented well the MP degradation rate data, as shown in Equation 2. Where: R is the overall conversion rate of MP; C MP is MP concentration; and k 1 is the first order kinetic constant.
The kinetic constant in the experiments without glucose was 0.063 ± 0.005 h -1 , and with glucose, 0.162 ± 0.014 h -1 .Therefore, we can state unequivocally that the addition of glucose increased the MP conversion rate.
The cellular production in the PFG reactors was around 80% (Figure 3), except for PFG6, which contained a higher concentration of MP.The PF reactors showed a decrease in the biomass production with a MP concentration that is increasing.Thus, the addition of glucose led to a different behavior than the one displayed by the reactors without glucose, indicating that, in the range of MP concentrations from 0.62 to 14.52 mg/L, the cellular growth was practically the same.
It is assumed that glucose can be indispensable both for the removal of MP and for cellular growth.However, it is necessary to apply statistical test to affirm the importance of the glucose addition for the MP removal.

Conclusions
MP concentrations of up to 60 mg/L were not toxic to Aspergillus niger AN400.The presence of a glucose concentration of 0.5 mg/L helped the removal of the pollutant.The highest MP removal rate achieved was 82% in the PFG1reactor, which was loaded with the lowest initial MP concentration of the group (0.062 mg/L).Therefore, the presence of glucose was indispensable for MP removal.
The first order kinetic model well-represented the rate of MP degradation, particularly in the reactors containing glucose.The kinetic constant in the experiments without added glucose was 0.063 ± 0.005 h -1 and in those with glucose, 0.162 ± 0.014 h -1 , indicating that the addition of glucose hastened the conversion of MP.
The cell growth in the PFG reactors was not affected by the increase in MP up to a concentration of 14.52 mg/L, but it was declined at an MP concentration of 24.89 mg/L.In the PF reactors, the cell growth decreased as the MP concentration increased.

A
kinetic model was adjusted to the Ro values as a function of the initial MP concentrations in the reactors.The Ro values were estimated, and the kinetic model was adjusted using Levenberg-Marquardt' s nonlinear regression method -Microcal Origin 5.0 ® (MARQUARDT, 1963).

CytochromeFigure 2 -
Figure 2 -Variation of MP concentration as a function of the reaction time in the PFG reactors.

Figure 1 -
Figure 1 -Variation of MP concentration as a function of the reaction time in the PF reactors.

Figure 3 -
Figure 3 -VSS profile in the PF and PFG reactors.

Table 1 -
Initial concentrations of MP used assays in batch reactors of controls, PF e PFG C: controls reactors; PF: reactors with fungi and methyl parathion; PFG: reactors containing fungi, glucose, and methyl parathion.

Table 2 -
Initial reaction rate (Ro) of MP degradation for reactors with (PFG) and without (PF) glucose * The reaction rate could not be determined based on data obtained under this condition.